Hydrogen peroxide targets the cysteine at the active site and irreversibly inactivates creatine kinase

In our study, we showed that at a relatively low concentration, H2O2 can irreversibly inactivate the human brain type of creatine kinase (HBCK) and that HBCK is inactivated in an H2O2 concentration-dependent manner. HBCK is completely inactivated when incubated with 2 mM H2O2 for 1 h (pH 8.0, 25 °C). Inactivation of HBCK is a two-stage process with a fast stage (k1 = 0.050 ± 0.002 min−1) and a slow (k2 = 0.022 ± 0.003 min−1) stage. HBCK inactivation by H2O2 was affected by pH and therefore we determined the pH profile of HBCK inactivation by H2O2. H2O2-induced inactivation could not be recovered by reducing agents such as dl-dithiothreitol, N-acetyl-l-cysteine, and l-glutathione reduced. When HBCK was treated with DTNB, an enzyme substrate that reacts specifically with active site cysteines, the enzyme became resistant to H2O2. HBCK binding to Mg2+ATP and creatine can also prevent H2O2 inactivation. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence data showed no tertiary structure changes after H2O2 treatment. The thiol group content of H2O2-treated HBCK was reduced by 13% (approximately 1 thiol group per HBCK dimer, theoretically). For further insight, we performed a simulation of HBCK and H2O2 docking that suggested the CYS283 residue could interact with H2O2. Considering these results and the asymmetrical structure of HBCK, we propose that H2O2 specifically targets the active site cysteine of HBCK to inactivate HBCK, but that substrate-bound HBCK is resistant to H2O2. Our findings suggest the existence of a previously unknown negative form of regulation of HBCK via reactive oxygen species.